DE19609479A1 - Endotoxin-specific membranes - Google Patents

Abstract

The invention concerns a microfiltration membrane for separating endotoxins from liquid media, in particular water, protein solutions or parenteralia. The microfiltration membrane is characterized by covalently bonded ligands for endotoxins, the ligands being carried by a polymer which is applied to the membrane.

Description

The invention relates to a microfiltration membrane for separation endotoxins from liquid media and their use this microfiltration membrane.

Endotoxins are lipopolysaccharides from the outer cell membrane Gram-negative bacteria that act as pyrogens. Due to the The ubiquity of bacteria is ubiquitous, too. However, unlike bacteria, you cannot use Stan Standard methods such as sterile filtration or autoclaving are removed or made harmless (1). For this reason it is sterile not synonymous with endotoxin-free. Is particularly critical the presence of endotoxins in injection or infusion solution solutions (parenterals) since they are already administered intravenously in Amounts of 1 ng per kg body weight have a febrile effect. The Symptoms are sufficient with a correspondingly high dosage (e.g. through large volume parenterals) to severe shock and death (2, 3). Therefore, almost all pharmacopoeias write next to the aseptic  strict endotoxin levels: e.g. B. 0.2 EU per mg Chloramphenicol for injection or just 0.003 EU per heparin unit (4). In practice, these requirements are met some difficulties. In particular, the production of organic Medicinal product cannot be endotoxin-free in all steps The main possible sources of endotoxin are:

• - Raw materials such as plasma or tissue that already contain bacteria can be mined.
• - In the case of recombinant products, the entry of host-spe specific endotoxin.
• - Bacterial contamination of devices, filters or auxiliary substances fen during manufacture.

The usual heat decontamination for thermostable active ingredients (30 minutes at 250 ° C) is also not ge for the preparations suitable as treatment with acids, bases or strongly oxidizing Agents (H₂O₂) (1).

When using activated carbon or depth filters such as ZETA PLUS there are often noticeable product losses, so that their application remains limited to water treatment (5, 6).

Ultrafiltration has a large population as a very gentle method achieved in the field of endotoxin removal. Here will i. a. worked with cut-offs of 10,000 or 5,000 to also effectively separate monomer components (MW approx. 14,000), which in addition to high molecular weight aggregates (up to several million Molecular weight). Nonetheless, there are always pros bleme with low molecular weight fragments, which also have a pyrogenic effect (e.g. Lipid A). This mainly affects hemo dialysis: although the dialysis buffers are ultrafiltered, develop e.g. B 400,000 hemodialysis patients annually in the United States septic symptoms (7). - The need for small cut-offs  also limits the use of ultrafiltration to De contamination of low molecular weight substances (8).

In the case of high molecular weight products such as pharmaceutical proteins, Albu Min supplements or heparin still exist in large quantities Difficulty: Endotoxin contamination occurs in these preparations only the possibility of repro to meet the requirements of FDA, USP or EP will.

In order to avoid this complex process, the product nevertheless, to lead to its purpose, the possibility was ge checks contaminated products via chromatographic sorbents to decontaminate selectively with endotoxin-specific ligands. This also did not bring the desired success: So far described bene affinity sorbents with His, Him, PMB as ligands despite high to very high association constants high endotoxin concentrations as unsuitable (9). In the presence of proteins, competing Pro Tin adsorption observed, leading to decreased endotoxin abrasion backup rates and sometimes high protein losses (especially with acidic proteins like BSA).

literature

(1) SK Sharma
Endotoxin detection and elimination in biotechnology Biotechnol. Appl. Biochem. 8: 5-22 (1986)
(2) N. Haeffner-Cavaillon, JM Cavaillon, L. Szabó Cellular receptors for endotoxin
Handbook of Endotoxins, Vol. 3: Biology of Endotoxins, 1-24
Elsevier Science Publishers BV (1985)
(3) DC Morrison, JL Ryan
Endotoxins and disease mechanisms
Ann. Rev: Med. 38 (1987), 417-32
(4) USP XXII Suppl. 5 (Nov. 1991)
(5) KC Hou, R. Zaniewski
Depyrogenation by endotoxin removal with positively charged depth filter cartridge
J. Parenteral Sci. Tech., Vol. 44, No. 4: 204-209 (1990)
(6) CUNO Newsletter for Pharmaceuticals (Oct. 1995), p. 3
(7) BP Smollich, D. Falkenhagen, J. Schneidewind, S. Mitzner, H. Klinkmann
Importance of endotoxins in high-flux dialysis
Nephrol. Dial. Transplant 3 (Suppl.) (1991) 83-85
(8) E. Flindt
Pyrogen removal using ultrafiltration
Memoscript CONCEPT symposium "Pyrogene II" (June 1983), pp. 54-60
(9) FB Anspach, O. Hillbeck
Removal of endotoxins by affinity sorbents
J. Chromatogr. A 711 (1995), 81-92

This prior art can be inventively by Microfiltration membrane for the separation of endotoxins from fluids media, especially water, protein solutions or parents Improve ralia, the microfiltration membrane by kova lent bound ligands for endotoxins, wherein the ligands are carried by a polymer based on the Membrane is applied.

For membrane technology and membrane production, Ho & Sirkar (Editor), Membrane Handbook, Verlag van Nostrand Reinhold, New York, 1992.

The covalently bound ligands can be

• (a) an endotoxin-specific ligand, preferably Histamine, histidine, polyethyleneimine, poly-L-lysine or poly myxin B and / or
• (b) act as a non-endotoxin-specific ligand per se, preferably diaminohexane, diethylaminoethyl or deoxycho lat.

In the membrane material for the microfiltra according to the invention tion membrane can be regenerated cellulose, cellulose act acet, polysulfone, polyethylene vinyl alcohol or polyamide, especially nylon.

In the case of the polymers, the microfiltra according to the invention tion membrane is applied, it can be a hydrophilic Act polymers, in particular dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethyl cellulose.

This hydrophilic polymer can be water-soluble, in what be swellable or water-insoluble.

The polymer can be removed from the microfiltration membrane according to the invention bran can be worn with the help of a spacer. The covalent too bound ligands can be carried by a spacer. At these spacers can be spacers that differ from Bisoxirane, glutaraldehyde, epihalohydrin or diisocyanate derive, if necessary after oxidative activation.

For activation and immobilization chemistry, which is also available on endo toxin-specific ligands and spacers, for example point to Hermanson, Mallia & Smith, Immobilized Affinity Ligand Techniques, Academic Press Inc., San Diego, 1992.

According to the invention, microfiltration membranes are provided, which are surface-modified in a suitable manner and remove endotoxins from water and aqueous solutions (buffers, protein solutions). The surface modification can consist in the application of a bifunctional covalently bonded spacer which is reacted with a hydrophilic polymer, non-specific interactions of the membrane, especially with proteins, being reduced. The covalently bound hydrophilic polymer can be reacted with endotoxin-specific ligands, if necessary via a further spacer. For the principle of surface modification of the membrane, see FIG. 1.

Endotoxin depletion success may vary in the presence of Proteins as dependent on the net charge of the proteins sen. By optimizing the conditions (pH value) acidic Proteins (such as BSA and mouse IgG 1) completely decontaminated without any significant loss of protein. In the case basic proteins (such as lysozyme and bFGF) high depletions are also achieved.

Polymer-coated microfiltration membranes according to the invention with covalently bound endotoxin-specific ligands can remove endotoxins in one pass, even from highly loaded solutions (6000 EU ml ^-1 ).

In principle, the membranes are built up according to Fig. 1. First, a hydrophilic polymer is applied via a spacer, which is then further, optionally via a spacer, reacted with endotoxin-specific ligands. The following are particularly suitable as membrane materials:

• - cellulose
• - polysulfone
• - PEVA (polyethylene vinyl alcohol)
• - polyamides (especially nylon, e.g. N66)

Reactive bifunctional compounds are suitable as spacers. Spe the following are suitable:

• Bisoxirane
• - glutardialdehyde
• - epihalohydrins
• - diisocyanates

To activate when using bisoxiran and epihalogen The vicinal diol bond formed in hydrine can possibly oxidize be applied by periodate, with an aldehyde group ent stands. The spacer bound to the membrane is further implemented with hydrophilic polymers. As such, the following are preferred:

• - dextran
• - polyvinyl alcohol (PVA)
• - Modified celluloses, especially hydroxyethyl cellulose (HEC)

The further reaction takes place either directly with the endotoxin specific ligands or again through the mediation of a the above-mentioned spacer, if necessary after its oxidative acti crossing. Act as endotoxin-specific ligands (see list of Abbreviations): DAH, Him, His, PEI, PLL, PMB. Even the more common have non-endotoxin-specific ligands such as DEAE and DOC proved to be highly specific in the membrane configuration at the same time high recovery of the proteins.

The performance of the developed membranes can be seen from the examples given. Endotoxin removal can almost always be said to be complete. It is usually below 1 EU ml ^-1 , often below the detection limit with the LAL test.

On control membranes (nylon without modification and with attached brought hydrophobic polymer with or without spacer) without endoto xin-specific ligands, no endotoxin depletion hold.

The new membranes can be used as an endotoxin Removal from water and parenterals. Even in the presence of Pro good results are achieved. In the case of basic pro However, one must take into account that interactions  of the proteins with the endotoxin that can lead to a endotoxic masking. Protein-bound endo toxin cannot be clearly demonstrated with the LAL test. In this context it should be mentioned that ge It is clear whether protein-bound endotoxin is still toxic.

The membranes of the invention can be used in a variety of ways the.

Medical-pharmaceutical area:

• - hemodialysis.
• - Safe infusion and injection solutions (Parenterals)
• - Safe diagnostics (e.g. antibodies).

In biotechnology:

• - Manufacture of pharmaceutical products.
• - Endotoxin depletion in process water and raw materials.
• - Decontamination of products (expenditure for processing ent falls)

Methods 1. Production of the membranes

Hydrophilic polymers, in particular dextran, polyvinyl alcohol and hydroxyethyl cellulose, are covalently bound to microfiltration membranes based on nylon (preferably 0.45 μm or larger). In the next step, endotoxin-specific ligands are immobilized on the applied polymers. Fig. 1 illustrates the construction of the membranes.

1.1. Membrane coating using the example of dextran

The nylon membranes were first activated with bisoxirane:
For this purpose, they were shaken for sixteen hours at 80 ° C. in a mixture of 9 ml bisoxirane, 1 ml ethanol and 1 ml 25 mM sodium carbonate buffer (pH 11) ( FIG. 2a). After thorough washing, each membrane with 5 ml of a 20 percent. Dextran 40,000 solution (pH 11) incubated for fifteen minutes at room temperature ( Fig. 2b). The membranes were then dried at 120 ° C. for 14 hours. To remove non-specifically bound dextran, the membranes were washed three times with 0.1 M sodium hydroxide solution and three more times with water.

As shown in Fig. 3, the coated membranes are characterized by significantly lower non-specific interactions - expressed by the amount of hemoglobin adsorbed.

From Fig. 3 also shows that with a simple dextran coating, the same effect can not be achieved as with PVA and HEC. A second layer can be used for further improvement, while a third layer has only minor effects. Dextran was therefore always used as a double coating.

1.2. Immobilization of endotoxin-specific ligands

The ligands PLL, PMB and PEI were either immobilized directly on the periodate-activated coating polymers or after incorporation of a periodate-oxidizable spacer (bisoxirane). The procedure is shown by way of example in FIG. 4. DEAE was coupled directly to the matrices without a spacer, the other never dermolecular ligands were bound via epibromohydrin.

1.2.1 PEI immobilization via bisoxirane

For activation, the membranes coated with hydrophilic polymers were incubated for three hours at room temperature in a mixture of 100 mg sodium borohydride, 5 ml bisoxirane and 45 ml 1 M sodium hydroxide solution. After hydrolysis of the free oxirane ring (30 minutes incubation at pH 2.5) and periodate oxidation of the vicinal diol obtained (90 minutes incubation in 0.2 M sodium periodate), the membranes were washed with a solution of 0.5 g PEI ( MW 50,000) in 0.1 M phosphate buffer, which was adjusted to pH 8, at room temperature, so that the structure shown in FIG. 1 results. Finally, it was washed with 1 M sodium chloride solution and water.

1.2.2. Histidine immobilization

Histidine was coated on epibromohydrin-activated via DAH Immobilized membranes. Epibromohydrin activation was like for Bisoxiran described performed. DAH was immobilized by reaction for 8 minutes with a mixture of 5 ml of Epibromhy activated in it and 5 ml of 4 M sodium hydroxide solution at 90 ° C and immediately at 80 ° C with L-histidine (0.5 g L-histidine in 20 ml Was water, pH 12). The finished membrane was covered with 1 M sodium chloride and washed water.

Corresponding regulations were applied for the coating with other polymers and the covalent bond with the other en dotoxin-specific ligands.

2. Depletion experiments

All studies on the adsorption behavior of endotoxins the membranes were run through at room temperature in dead-end mode guided.

One membrane disk was placed on the bottom of an ultrafiltration cell (membrane area 13.4 cm²) fixed and with 30% ethanoli 0.1 M sodium hydroxide solution, 1.5 M sodium chloride solution and pyro GM free water washed to remove traces of endotoxin. After equilibration of the membrane, 20 ml each were contaminated nated solution at a flow rate of 2 ml / min the membrane is filtered. The filtrate was collected and in the LAL test examined.  

3. Endotoxin test

For endotoxin quantification in the starting solutions and A chromogenic Limulus amebocyte lysate test was used to filter the filtrates (LAL test) used. This test is based on endotoxin induces the release of the chromogen p-nitroaniline, whereby between the released amount of p-nitroaniline and the present endotoxin concentration in the range 0 to 1.2 EU / ml linear relationship exists. From the photometric p-nitro Aniline determination can be done using a calibration line (Standard endotoxin E. coli 0111: B4) on the endotoxin concentrations tration of the samples can be closed.

The LAL test was launched in Europe in 1985 by the European Medicines book commission for testing for endotoxins and he introduced has been using water for injections in the monograph since 1989 purpose "the rabbit test.

Examples of use

1. ( Fig. 5) depletion from highly loaded buffer solutions Feed: 20 ml 20 mM phosphate buffer (pH 7) mixed with 6000 EU / ml
The membranes marked with -d represent membranes in which the incorporation of a spacer has been dispensed with.

2. ( Fig. 6 to 7) depletion from endotoxin-enriched BSA solutions
Feed: 20 ml 20 mM phosphate buffer (pH 4.66) mixed with 1 mg / ml BSA and 6610 EU / ml
Protein recovery BSA

3. ( Fig. 8) depletion from commercial BSA
Feed: 20 ml 20 mM phosphate buffer (pH 4.66) with 1 mg / ml BSA
Endotoxin concentration 65 EU / ml

4. ( Fig. 9 to 10) depletion from commercial lysozyme
Feed: 20 ml 20 mM phosphate buffer (pH 7) with 1 mg / ml lysozyme endotoxin concentration 134 EU / ml
Protein recovery lysozyme

5. ( Fig. 11 to 12) depletion from MAX 16 H 5
Feed: 20 ml 20 mM phosphate buffer (pH 5.5) with 3 mg / ml protein
Endotoxin concentration 62.5 EU / ml
Protein recovery IgG

6. Depletion from already purified bFGF
Feed: 5 ml bFGF, which contained 9 EU / ml
The depletion process of a PEI membrane was examined. 0.202 EU / ml was still detectable in the filtrate.

7. Depletion from Milli-Q water that has been treated with endotoxin
Feed: 1 l water mixed with 270 EU / ml
A PEI and a DAHHis membrane were used for depletion.
PEI filtrate: <0.015 EU / ml
DAHHis filtrate: 0.07 EU / ml

used abbreviations

BSA bovine serum albumin
bFGF basic fibroblast growth factor
DAH diaminohexane
DEAE diethylaminoethyl
DEX dextran
DEX / 2 is a membrane that has been coated twice in succession with dextran
DEX / 3 is a membrane that has been coated three times in a row with dextran
DOC deoxycholate
EP European Pharmacopoeia
EU endotoxin unit
FDA Food and drug administration
HEC hydroxyethyl cellulose
Him histamine
His histidine
MW molecular weight
N66 untreated nylon membrane
PEI polyethyleneimine
PLL poly-L-lysine
PMB polymyxin B
PVA polyvinyl alcohol
USP US Pharmacopoeia

Claims (10)

1. microfiltration membrane for the separation of endotoxins from liquid media, in particular water, protein solutions or parenterals, characterized by covalently bound ligands for endotoxins, the ligands being carried by a polymer which is applied to the membrane. 2. microfiltration membrane according to claim 1, characterized by

• (a) an endotoxin-specific ligand, preferably histamine, histidine, polyethyleneimine, poly-L-lysine or polymyxin B and / or
• (b) a non-endotoxin-specific ligand per se, preferably diaminohexane, diethylaminoethyl or deoxycholate.

3. microfiltration membrane according to claim 1 or 2, characterized net by regenerated cellulose, cellulose acetate, polysulfone, Polyethylene vinyl alcohol or polyamide, especially nylon as Membrane material. 4. microfiltration membrane according to one of the preceding claims che, characterized by a hydrophilic polymer, in particular Dextran, polyvinyl alcohol or modified cellulose preferably hydroxyethyl cellulose. 5. microfiltration membrane according to claim 4, characterized net that the hydrophilic polymer itself water soluble or is insoluble in water. 6. microfiltration membrane according to one of the preceding claims che, characterized in that the polymer from the membrane is worn with the help of a spacer. 7. microfiltration membrane according to claim 6, characterized by one of bisoxirane, glutardialdehyde, epihalohydrin or Diisocyanate-derived spacer. 8. microfiltration membrane according to one of the preceding claims che, characterized in that the ligands of the polymer be worn with the help of a spacer. 9. microfiltration membrane according to claim 8, characterized by one of bisoxirane, glutardialdehyde, epihalohydrin or Spacer derived from diisocyanate, optionally after oxidative Activation.   10. Use of a microfiltration membrane according to one of the preceding claims for removing endotoxins from fluids sigen media, especially from water, protein solutions or Parenterals.